Cyclo(His-Pro) is believed to be a metabolite of TRH. A specific antiserum directed against cyclo(His-Pro) was used to detect immunoreactive material in human serum. Gel filtration column chromatography was used to establish that cyclo (His-Pro)-like immunoreactivity was found not only in a free form established to be identical to cyclo(His-Pro) by high pressure liquid chromatography analysis on reverse phase and cation exchange columns, but also in a fraction of about 100,000 mol. wt. Free cyclo(His-Pro) was released by heating column fractions of 70,000 mol. wt. that were not immunoreactive before heating. A procedure for estimation of the content of free cyclo(His-Pro) in human serum was developed. Normal levels of cyclo(His-Pro) determined by this procedure were in the range of 11-33 pmol/ml. The level of cyclo(His-Pro) in sera of individuals with hypothyroidism, hyperthyroidism, or alcoholic cirrhosis was in the normal range while patients with renal failure had approximately 3-fold elevated levels of the peptide. Analysis of human sera drawn at 3 hr. intervals over a 24 hr period suggested a circadian rhythm of cyclo(His-Pro) levels.